Guide to Research Techniques in Neuroscience

Guide to Research Techniques in Neuroscience

Matt Carter

Jennifer C. Shieh

Stanford University, School of Medicine, Stanford

Table of Contents

Cover image

Title page

Copyright

Preface

Foreword

Introduction

Levels of Investigation

Methods of Studying the Nervous System

Understanding Techniques in Neuroscience

Chapter 1. Whole Brain Imaging

Publisher Summary

Structural Brain Imaging Techniques

Functional Brain Imaging Techniques

Functional Imaging Experimental Design and Analysis

Conclusion

Suggested Reading and References

Chapter 2. Animal Behavior

Publisher Summary

Considerations for Choosing and Performing a Behavioral Assay

Rodent Behavioral Paradigms

Drosophila Behavioral Paradigms

C. Elegans Behavioral Paradigms

Nonhuman Primate Behavioral Paradigms

Conclusion

Suggested Reading and References

Chapter 3. Stereotaxic Surgeries and In Vivo Techniques

Publisher Summary

Stereotaxic Surgeries

Implants for Long-Term Access to the Brain

Measuring Neural Activity in Vivo

Measuring Neurochemistry in Vivo

Manipulating the Brain in Vivo

Conclusion

Suggested Reading and References

Chapter 4. Electrophysiology

Publisher Summary

A Brief Review of the Electrical Properties of Neurons

The Electrophysiology Rig

Types of Electrophysiology Recordings

Electrophysiology Tissue Preparations

Methods of Manipulating Neurons During Electrophysiology Experiments

Conclusion

Suggested Reading and References

Chapter 5. Microscopy

Publisher Summary

Essential Principles of Microscopy

Light Microscopy

Fluorescence Microscopy

Electron Microscopy

Preparing and Interpreting Microscopy Data

Conclusion

Suggested Reading and References

Chapter 6. Visualizing Nervous System Structure

Publisher Summary

Tissue Preparation

Visualizing Morphology

Visualizing Gene and Protein Expression

Visualizing Circuitry

Conclusion

Suggested Reading and References

Chapter 7. Visualizing Nervous System Function

Publisher Summary

Static Markers of Activity

Visualizing Neural Activity

Optically Manipulating Neural Activity

Visualizing Protein Function

Optically Manipulating Protein Function

Conclusion

Suggested Reading and References

Chapter 8. Identifying Genes and Proteins of Interest

Publisher Summary

A Brief Review of Genes and Proteins

Genetic Screens

In Silico Screens

Blast

Molecular Screens

Conclusion

Suggested Reading and References

Chapter 9. Molecular Cloning and Recombinant DNA Technology

Publisher Summary

Isolating DNA Fragments

Cloning DNA

Purifying DNA

Identifying DNA

Conclusion

Suggested Reading and References

Chapter 10. Gene Delivery Strategies

Publisher Summary

Physical Gene Delivery

Chemical Gene Delivery

Viral Gene Delivery

Conclusion

Suggested Reading and References

Chapter 11. Making and Using Transgenic Organisms

Publisher Summary

Transgenes

The Transgenic Construct

Binary Transgenic Systems

Making Transgenic Organisms

Conclusion

Suggested Reading and References

Chapter 12. Manipulating Endogenous Genes

Publisher Summary

Genetic Targeting

Disrupting Gene Products

Conclusion

Suggested Reading and References

Chapter 13. Cell Culture Techniques

Publisher Summary

Cell Culture Equipment and Reagents

Immortalized Cell Lines

Primary Cell and Tissue Culture

Stem Cell Cultures

Manipulating Cells in Culture

Conclusion

Suggested Reading and References

Chapter 14. Biochemical Assays and Intracellular Signaling

Publisher Summary

Introduction to Signal Transduction and Intracellular Signaling

Fundamental Tools for Studying Proteins

Investigating Protein Expression

Investigating Protein-Protein Interactions

Investigating Posttranslational Modifications

Investigating Protein-DNA Interactions

Conclusion

Suggested Reading and References

Glossary

Index

Copyright

Academic Press is an imprint of Elsevier

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Cover image of fly brain used with generous permission from Liqun Luo and Chris Potter, Dept of Biology, Stanford University

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Library of Congress Cataloging-in-Publication Data

Carter, Matt, 1978-

Guide to research techniques in neuroscience / Matt Carter, Jennifer C. Shieh.

p.; cm.

Includes bibliographical references.

ISBN 978-0-12-374849-2

1. Neurosciences—Research—Methodology. I. Shieh, Jennifer C., 1981- II. Title. [DNLM: 1. Nervous System Physiological Phenomena. 2. Diagnostic Imaging—methods. 3. Laboratory Techniques and Procedures. WL 102 C324g 2010]

RC337.C37 2010

616.85'20072—dc22

2009021267

British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library.

ISBN: 978-0-12-374849-2

For information on all Academic Press publications visit our Web site at www.elsevierdirect.com

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Preface

Matt Carter

Jennifer C. Shieh

Stanford, California, 2009

The story of why we decided to write this book can essentially be summarized as: we tried to find a book just like it, couldn’t find one anywhere, and ultimately decided the book would be so useful that we would write it ourselves.

When we were advanced undergraduates and beginning graduate students, we found the number of techniques used in neuroscience research daunting. In no other biological field are students required to learn biochemistry, molecular biology, genetics, electrophysiology, microscopy, histology, behavioral assays, and human imaging technologies, not to mention the basic biological and physical properties that allow these methodologies to work. It is true that each neuroscientist only practices a handful of these general techniques. However, all neuroscientists must learn to understand and appreciate one another’s research, even if they don’t perform the specific experiments themselves.

For example, consider the study of the auditory system. Auditory research can be performed in humans using functional magnetic resonance imaging (fMRI) and other methods of whole-brain imaging. Auditory research can also be performed in animal models, from standard laboratory animals (mice, rats, flies) to animals that have unique auditory properties that make them particularly well suited for auditory research (bats, barn owls). These animal models can be studied using in vivo electrophysiology while they are anesthetized or while performing behavioral tasks. Additionally, the cells responsible for hearing and interpreting auditory stimuli can be examined in vitro—for example, by recording electrical signals from isolated hair cells. Histological and biochemical techniques can highlight the specific ion channels and other proteins that provide auditory cells their physiological properties. Genetic methods can be used to investigate the role of molecules that make hearing possible or cause auditory deficits. Thus, anyone interested in the auditory system, or any other specific subfield of neuroscience, must be prepared to read about and analyze the contributions of studies using a wide spectrum of techniques.

As students new to neuroscience research, we were overwhelmed with the expectation that we understand all of these methods. Moreover, we were surprised by the number of advanced students who seemed to pretend that they understood all these techniques when they really only understood the specific techniques they used in their own studies. Sometimes, these students, and even some postdocs and faculty, would confess to us, I should have learned that long ago, but I just don’t really understand it. We would hear scientists who use electrophysiology and fMRI techniques sometimes say, Who cares about a blob on a gel? Likewise, we would hear geneticists and molecular biologists confess, I don’t know what the squiggly lines in electrophysiology studies mean, or I don’t know why fMRI experiments are so hard—can’t you just put someone in a scanner and turn on the machine? In short, it became obvious that there should be a guide to neuroscience techniques. So we tried to find one.

Finding a guide to neuroscience techniques turned out to be more difficult than we imagined. During our first trips to the annual Society for Neuroscience conference, we tried to find a simple techniques book among the hundreds of books being promoted by different publishers. Amazingly, there was no single book that aimed to explain the breadth of neuroscience techniques at a basic level. There were detailed books written about single techniques (PCR Methods or Making Transgenic Animals) and recipe books filled with protocols, but no book addressed techniques as a subject themselves. Even the almighty Internet did not provide thorough insight into standard techniques. On the Internet, you can find protocols for doing a western blot and learn that it is a technique that measures the amount of protein in a sample. But you cannot find information about other ways a scientist can measure protein expression or why you might choose a western blot over these other methods. There are no example figures from the literature, nor are there suggestions of what to look for in these figures. Our dream book would contain a description of western blots, a comparison of western blots with similar methods, and an example of what the data looked like in the literature.

No such book existed, and we wanted one. With the encouragement of faculty members and students at Stanford, we decided to write this book. We also decided that the information would make an excellent seminar course. Along with a talented student colleague of ours, Saul Villeda, we created a 9-week techniques course called Understanding Techniques in Neuroscience. This class surveys neuroscience techniques and provides examples from the literature. We wrote a 110+-page course reader to accompany the class and quickly decided that this text should also form the basis of a future book that could exist independently of the class.

The class was amazingly well received. The first year, our class had 15 students. Through word-of-mouth, the class doubled in size the next year. The third year, over 100 students attended our lectures, including undergraduates, graduate students, postdocs, and occasionally a faculty member. We believe the number of people taking the course demonstrates that neuroscientists are interested in learning about one another’s methods, as well as the need for formalized education about neuroscience techniques.

Therefore, we decided to adapt our course reader into this book. It is the book that we so desperately wanted when we were beginning neuroscience students, and we are happy that it is now possible to find at Society for Neuroscience meetings. We learned a tremendous amount of information while researching and writing this book, and we hope that you find it helpful for your own education.

We would like to first thank the editorial staff at Elsevier/Academic Press for making this book a reality. From Susan Lee, who initially saw the potential of our course reader, to Melissa Turner and Mica Haley, who managed to get the final product with only minor hair-pulling.

This book would not be possible without the guidance and help of the incredible faculty at Stanford. In particular, Bill Newsome provided tremendous encouragement and support throughout the writing process. As promised, he will receive a fine bottle of Woodford Reserve upon the successful publication of this book. We must also acknowledge the understanding of our research advisors Luis de Lecea and Sue McConnell. As promised, they will receive our theses without further delay. Finally, we thank Mehrdad Shamloo and Mehrdad Faizi at Stanford’s Behavioral and Functional Neuroscience Laboratory and Jon Mulholland and Lydia-Marie Joubert of the Cell Sciences Imaging Facility for their expert advice.

Our teaching partner Saul Villeda researched many of the methods that are detailed in this book and helped prepare the original course reader. Saul is an amazing teacher, and it has been our pleasure to work with him over the years, both as colleagues and friends.

We also received substantial guidance and support from the Stanford neuroscience community. So many students and postdocs spent their valuable time and energy to help us understand techniques, edit our chapters, and provide encouragement as we finished the book. Special thanks to Raag Airan, Björn Brembs, Brittany Burrows, Laurie Burns, Kelsey Clark, Emily Drabant, Mary Hynes, Pushkar Joshi, Rachel Kalmar, Jocelyn Krey, Dino Leone, Jie Li, Scott Owen, Georgia Panagiotakos, Chris Potter, Elizabeth Race, Victoria Rafalski, Andreas Rauschecker, Magali Rowan, Rory Sayres, Bob Schafer, Jana Schaich Borg, John Wilson, and Sandra Wilson, who provided substantial suggestions to improve the content and text. We completed this book with our neuroscience colleagues in mind, and we hope that all future neuroscience students find this book helpful to their education and throughout their careers.

Finally, we thank our significant others, Vishal Srivastava and Alison Cross. Thank you so much for giving us time, love, and encouragement.

Foreword

William T. Newsome, Ph.D.

Professor, Department of Neurobiology, Stanford University, Investigator, Howard Hughes Medical Institute

Understanding how the brain works is perhaps the greatest challenge facing contemporary science. How mental life is rooted in the biology of the brain is intrinsically fascinating, and researchers from remarkably diverse scientific backgrounds are being drawn increasingly to the field of neuroscience. Psychologists, molecular biologists, physiologists, physicists, engineers, computer scientists, and more are all contributing importantly to the richness of modern neuroscience research. The brain remains the most complex and enigmatic entity in the known universe, and all relevant scientific techniques and perspectives must ultimately be brought to bear to solve its mysteries.

The quickening pace and diversity of neuroscience research pose substantial challenges, however, for new students and for established researchers seeking to enter the field for the first time. The techniques used to measure and manipulate the nervous system are dizzying in their scope and complexity, and some of the important new approaches are accompanied by clouds of jargon that are all but impenetrable to outsiders. Fortunately for those of us who are perplexed, a concise, no-nonsense guidebook has now arrived. The Guide to Research Techniques in Neuroscience presents the central experimental techniques in contemporary neuroscience in a highly readable form. It is all there—from brain imaging to electrophysiology to microscopy to transgenic technologies. Matt Carter and Jennifer Shieh take us on a very practical cook’s tour of research techniques, all the while providing concise overviews of exactly where and how particular techniques fit into the grand scheme of the basic questions being asked of the nervous system.

As a neurophysiologist, I read quickly through the electrophysiology and brain imaging chapters, noting with appreciation the efficient presentation of basic information that should be in the intellectual repertoire of any aspiring neuroscientist. But I read avidly the chapters on cloning, gene delivery, and use of transgenic animals—the exotica (to me!) of molecular biological approaches made clean, simple, and pleasingly devoid of specialist vocabulary. I rather suspect that most neuroscience professionals will have the same experience reading this book, encountering high-yield veins of useful information interspersed with much they already know. For the student or postdoc just entering the field, however, the vast majority of the book will be very high-yield.

This book is an essential resource for anyone—from the beginning graduate student to the seasoned faculty member—who could use an efficient guidebook at his or her side while reading papers written by any of their colleagues, irrespective of the level of analysis, from small molecules to spike trains. It may not render the mysterious raster plots of systems lectures immediately clear, but it will orient you to the key types of information you want to take home from that talk. This book is a little gem! Read it, rely on it, and pass it on to others.

Introduction

The human mind has been studied for thousands of years, but the human brain, as well as the brains of other species, has only been studied for about a century. Only 150 years ago, the ability to study the nervous systems of humans and other animals was limited to direct observation and by examining the effects of brain damage in people and other organisms. With the advent of histology came the ability to visualize and differentiate between neurons based on morphology. The great neuroscientist Santiago Ramón y Cajal used a method called Golgi staining to visualize the morphology and architecture of neurons and their circuits throughout the brain. Cajal used the Golgi stain to propel the field of neuroscience into its modern state.

In the history of neuroscience, each leap forward in knowledge has been based on a leap forward in techniques and technology. Just as Ramón y Cajal used Golgi staining to greatly advance our understanding of the structure of the nervous system, scientists throughout the twentieth century used more and more advanced techniques to contribute to our understanding of the function of the nervous system: Eccles, Hodgkin, and Huxley used intracellular recording technology to investigate the ionic basis of membrane potentials; Hubel and Wiesel used extracellular recording technology to investigate how information is processed and recorded in the visual system; Neher and Sakmann used patch-clamp technology to investigate the physiology of single ion channels. In the latter half of the twentieth century, the explosion of molecular biology techniques and methods of genetically manipulating model organisms allowed neuroscientists to study individual genes, proteins, and cell types. Technology has progressed so far in the past 100 years that the Golgi stain itself seems to have been reinvented through powerful technologies (Chapter 11) that allow investigators to turn specific neurons different colors to further investigate the structure and connectivity of the nervous system.

The modern neuroscientist now has hundreds of techniques that can be used to answer specific scientific questions. This book contains 14 chapters that provide an overview of the most commonly used techniques. Although there are dozens of techniques that seem very different at first glance, many of them attempt to study the nervous system in the same way. For example, transcranial magnetic stimulation (Chapter 1), physical lesions (Chapter 3), pharmacological inhibition (Chapter 3), optogenetic inhibition (Chapter 7), and genetic knockdown or knockouts (Chapter 12) are all attempts to test the effect of a loss-of-function of some aspect of the nervous system on another aspect of the nervous system. For each level of investigation (whole brains to individual genes), research strategies can be similar even if the techniques used are very different.

Levels of Investigation

Something immediately obvious to all students of neuroscience is that the nervous system is exceptionally complicated and can be examined at multiple levels of investigation. The basic functional unit of the nervous system is the neuron. The human brain is composed of approximately 100 billion neurons that are connected into circuits via approximately 100 trillion synapses. Neural circuits are organized into anatomical structures and larger networks of neurons that can integrate information across modalities from many different parts of the brain. These networks process sensory information from the external and internal environment and provide the neural basis of cognition—learning, memory, perception, decision making, emotion, and other higher-order processes. The final output of the nervous system is a behavior composed of a coordinated motor action. This behavior can either be extremely simple, such as a motor reflex, or incredibly complicated, such as dancing, typing, or playing a musical instrument. Behavior is usually defined not just by what an organism does, but what it chooses to do. Therefore, except in rare circumstances of lesion or disease, cognition and behavior are inseparably linked, and in animals other than humans, behavior is used as a read-out of animal cognition.

Just as one can start with a neuron and scale up toward circuits, cognition, and behavior, a scientist can also scale down and examine the components that make up a neuron. A neuron is itself defined as having a cell body (soma), axon, and dendrites. These neuronal components contain subcellular specializations that make the neuron unique among other cell types. Specialized organelles in a neuron, such as vesicles containing neurotransmitters, provide the cell with the ability to signal to other neurons. Specialized cytoskeleton processes allow a neural process to extend great distances throughout the brain and body. Several proteins provide neurons with their intercellular signaling abilities and physiological characteristics. For example, biosynthetic enzymes produce neurotransmitters, while other proteins serve as receptors for these signaling molecules. One of the most important types of proteins in the nervous system form ion channels, the transmembrane structures that allow neurons to become electrically active under certain conditions. All of these proteins are the products of genes, the functional units of an organism’s genome. The human genome contains approximately 30,000 genes, with each neural subtype expressing its own subset of these genes.

The complexity of the nervous system is awesome in scope. It is amazing that a mutation in a single gene, such as a gene that codes for a transmembrane ion channel, can produce effects that alter the electrical properties of a neuron, in turn altering the normal firing patterns of a neural circuit and thus causing an abnormal behavior.

A neuroscientist can approach the study of the nervous system through any of these levels of organization. The 14 chapters of this book provide a guide to the types of experiments that can be performed at each level. However, irrespective of technique, the basic scientific approach one can use to study the nervous system is consistent from level to level, whether the subject is human cognition or axon guidance in cell culture. Next we will examine the basic approaches to designing experiments in the nervous system.

Methods of Studying the Nervous System

There are four general methods of studying the nervous system: (1) examining case studies—identifying interesting events that have occurred naturally and using these events to develop hypotheses that can be tested in future experiments; (2) screens—searching for anatomical structures, neurons, proteins, or genes that could play a role in a subject of interest; (3) description—using techniques that allow a scientist to observe the nervous system without manipulating any variables; and (4) manipulation—testing hypotheses by determining the effect of an independent variable on a dependent variable. Each of these four methods is described in detail here.

Examining Case Studies

A case study is an example of an event that happened to a subject (most often a human or group of humans) that demonstrates an important role for an aspect of the nervous system. The circumstances surrounding the event are usually nonrepeatable and cannot be precisely recreated in a laboratory setting. Such demonstrations are, therefore, not true experiments in that no variables are deliberately controlled by a scientist. However, these events can often reveal substantial information about an aspect of neural function that was previously unknown.

For example, consider the case of Phineas Gage, a railroad worker who was involved in an accident in 1848 that caused an iron rod to pass through his skull. The rod entered the left side of his face, passed just behind his left eye, and exited through the top of his head, completely lesioning his frontal lobes. This is an amazing event, not only because Gage survived (and lived for another 12 years), but also because it informed scientists about the function of the frontal lobe of the brain. The event allowed investigators to retrospectively ask the question What is the effect of removing the frontal lobe on consciousness and behavior? According to Gage’s friends, family, and co-workers, he was no longer Gage. He retained the ability to learn, remember, sense, and perceive his environment, to execute motor functions, and to live a fairly normal life, but it seemed to people who knew him that his personality had changed completely. After the accident, Gage was less polite, erratic, unreliable, and offensive to others. He wound up losing his job at the railroad, not because of any physical or mental incapacity, but because he was simply so disrespectful and offensive that people could not stand to work with him.

This case study is not a true experiment; no scientist decided to test the removal of the frontal lobe on personality. But the incident, and others like it, allows neuroscientists to form hypotheses based on naturally occurring events. Because of Gage’s story, neuroscientists could hypothesize about the contribution of the frontal lobe to human personality. Future experiments could test these hypotheses on animal models (that share certain human personality traits) and even attempt to identify neural circuits that contribute to human behaviors.

Screens

A screen is a method that allows an investigator to determine what nuclei, neurons, or genes/proteins may be involved in a particular biological process. Such experiments are not necessarily driven by a hypothesis, but the experiment identifies candidates that can form the basis for future hypothesis-driven research. For example, a neuroscientist who wants to identify genes involved in body weight regulation may compare gene expression profiles in central feeding centers of the brain in both fed and starved animals: genes expressed in starved animals relative to fed animals may be important for generating the motivation to eat.

Screens can be performed at multiple levels of investigation. When a cognitive neuroscientist places a human subject into an fMRI scanner and examines which brain areas show increased activation in response to a specific stimulus or task, the scientist is essentially performing a screen of different brain regions. When a fly geneticist examines thousands of mutagenized flies for deficiency in a behavioral task, the scientist is attempting to identify genes necessary for that behavior to occur. Such genes can then be tested in future experiments. Thus, screens can be performed to identify interesting molecules or entire brain regions.

Description

Descriptive science is the act of simply observing properties of the nervous system without manipulation. This type of research is usually the first step in acquiring knowledge about a newly discovered gene, protein, or neuronal subtype. For example, an investigator could describe the sequence of a gene and where in the brain the gene is expressed. Likewise, in the case of proteins, an investigator could describe the amino acid sequence of the protein and where in the brain the protein is expressed. Neurons can be described in terms of what genes/proteins they express, their morphology, how many neurons make up a population of neurons, and their electrophysiological properties.

It is important to note that just because a study may be descriptive does not mean that it is necessarily easier than other types of experiments. Observation and description form the foundation for understanding the relationship between structure and function, as well as providing insight about what elements to manipulate in future experiments. Thus, descriptive neuroscience plays just as important a role in modern research as when Ramón y Cajal observed the structure of neurons 100 years ago.

Manipulation

Manipulating an aspect of the nervous system or environment and examining the effect this perturbation has on a separate aspect of the nervous system is the only way to test a hypothesis in neuroscience. A manipulation experiment tests the effect of X on Y. The variable that is manipulated, X, is referred to as the independent variable. The part of the system that is measured, Y, is referred to as the dependent variable.

Two of the most common types of manipulation experiments are loss-of-function and gain-of-function experiments. In a loss-of-function (also called "necessity") experiment, a part of the nervous system is diminished or removed in an attempt to determine if it is necessary for a certain process to occur. The following questions are all loss-of-function questions:

 Is a normal copy of the gene Fezf2 necessary for the proper development of the cerebral cortex?

 Is the receptor for the hypocretin neuropeptide necessary for normal sleep/wake transitions in mammals?

 Is electrical activity in the medial geniculate nucleus required for auditory-driven spikes in auditory cortex?

 Can human patients with damage to the cerebellar vermis perform as well as healthy controls on a verbal-memory task?

In all of these experiments, an aspect of the nervous system is partially or totally disrupted, whether it is a gene, a protein, electrical activity, or an entire brain structure. The independent variable is the loss of the structure, and the dependent variable is the effect on another aspect of the nervous system. Sometimes, a good follow-up for a loss-of-function experiment is a rescue experiment in which the aspect of the nervous system that is lost is deliberately returned. For example, if it is found that a certain line of fruit flies lacks a gene that is necessary for proper development of an eye, the specific gene can be reintroduced to the flies in transgenic experiments to see if the functional gene can rescue the aberrant phenotype.

In a gain-of-function (also call "sufficiency") experiment, an aspect of the nervous system is increased relative to normal. This may include an increased expression of a gene or protein, an increase in electrical activity in a brain region, or an increase of a particular neurotransmitter in the extracellular medium. The aspect of the nervous system that is increased is the independent variable, and the effect on another part of the nervous system is the dependent variable. The following questions are all gain-of-function questions:

 Can an increase in the gene TrpA1 cause mice to be hypersensitive to cold temperatures?

 Can an introcerebroventricular injection of Neuropeptide Y cause an increase in feeding behavior in rats?

 Can electrical microstimulation of the lateral geniculate nucleus increase spike frequencies over time in area V4 of visual cortex?

 Can stimulation of the motor cortex in human subjects using transcranial magnetic stimulation (TMS) cause motor behaviors?

In both loss-of-function and gain-of-function experiments, it is important not to overstate the conclusions of the experiments. For example, consider a loss-of-function experiment in which a mouse that lacks a gene is unresponsive to painful stimuli. The investigator could conclude that this gene is necessary for proper performance on an assay for pain detection. However, an inappropriate conclusion would be that this gene regulates pain detection. Perhaps this gene is responsible for normal development of the spinal cord and the mouse lacks all peripheral sensation. Alternatively, this gene may code for a protein that is necessary for normal development of the thalamus; if improper development of the part of the thalamus that receives information about painful stimuli causes the stimuli not to reach somatosensory cortex, the animal will not perform normally on the pain-detection task. Careful controls are necessary to reach appropriate conclusions.

Understanding Techniques in Neuroscience

We hope that these 14 chapters serve as a useful guide to studying the nervous system. There are two important points to keep in mind while reading these chapters: (1) All of the techniques described throughout this book depend on the principles just mentioned. For each level of investigation that a scientist may choose to study, the same four general types of methods exist: examining case studies, screens, description, and manipulation. The same principles that are used to study brain activity in awake, human subjects are used to study genes and proteins in tissue samples. The methods may vary, but the principles remain the same. (2) It is also important to remember that techniques and methods should never be the guiding force behind doing experiments in research. Ideally, experiments should be performed in order to answer an interesting question, not the other way around. A technique should not be used for its own sake but because it is the best technique available to answer a particular research question. Therefore, we hope this book answers your questions about what techniques are available in modern neuroscience research and answers your specific research questions as well!

Chapter 1

Whole Brain Imaging

Publisher Summary

The purpose of this chapter is to explain the brain imaging technology and provide insights into how experiments are designed and interpreted. Whole brain imaging technology can essentially be divided into two categories: structural and functional. Structural techniques produce images of the anatomical architecture of the brain, whereas functional techniques produce images of the physiological processes that underscore neural activity. This discussion surveys both classification of techniques and describes how they can be used in modern neuroscience research. Further, it focuses on magnetic resonance imaging (MRI) and functional magnetic resonance imaging (fMRI). MRI takes advantage of the magnetic properties of neural tissue to produce an image. Most often, MRI utilizes the magnetic properties of hydrogen protons, as they are highly abundant in the fluids and organic compounds of the brain and body. The main function of an MRI scanner is to artificially excite these hydrogen protons and then measure their relaxation properties over time. FMRI technology takes advantage of the signal intensity of another substance within the brain: hemoglobin, the protein in the blood that carries oxygen to cells. On a T2 image, oxyhemoglobin (the oxygen-carrying form of hemoglobin) has a relatively stronger magnetic resonance signal than deoxyhemoglobin. Thus, fMRI allows an investigator the opportunity to examine changes in the oxygenation-state of hemoglobin over time. Additionally, the chapter surveys the essential components of a functional imaging experiment: forming hypotheses, choosing appropriate task paradigms, performing experiments, acquiring and analyzing data, and producing figures for publication.

After reading this chapter, you should be able to:

 Compare the relative strengths and limitations of different structural and functional brain imaging techniques

 Explain the physical and physiological basis of MRI/fMRI technology

 Describe the components of functional brain imaging experimental design: formulating a hypothesis, choosing task paradigms, performing the experiment, acquiring and analyzing data, and constructing figures

Techniques Covered:

 Structural techniques: cerebral angiography, computerized tomography (CT), magnetic resonance imaging (MRI), diffusion MR imaging

 Functional techniques: functional magnetic resonance imaging (fMRI), positron emission tomography (PET), single-proton emission computerized tomography (SPECT), electroencephalography (EEG), magnetoencephalography (MEG), optical imaging

 Techniques used to investigate the necessity and sufficiency of a specific brain region for a cognitive function: transcranial magnetic stimulation (TMS) and case studies

Modern brain imaging technology can seem like magic. The ability to produce detailed images of the human brain without physically penetrating the skull is a technological marvel that has saved thousands of lives and allowed scientists to study the structure of the brain throughout development, disease, and aging. Furthermore, the ability to image neural activity in the brain during cognition has provided scientists the opportunity to correlate activity in distinct brain regions with specific mental operations, a truly remarkable achievement. Indeed, colorful figures depicting activity in the human brain dazzle scientists and nonscientists alike.

Of course, brain imaging technology is not magic. The technology that produces detailed images of the brain depends on complex physics, expensive equipment, and skilled technicians. As with all scientific experiments, brain imaging studies must be well designed, the data accurately analyzed, and the results carefully interpreted. The purpose of this chapter is to explain the ostensible magic of whole brain imaging technology and provide insight into how experiments are designed and interpreted.

Whole brain imaging technology can essentially be divided into two categories: structural and functional. Structural techniques produce images of the anatomical architecture of the brain, whereas functional techniques produce images of the physiological processes that underscore neural activity. This chapter will survey both classifications of techniques and describe how they can be used in modern neuroscience research. We will focus on MRI and fMRI technology due to the widespread use of these techniques in the neuroscience literature. After reviewing these techniques, we will survey the essential components of a functional imaging experiment: forming hypotheses, choosing appropriate task paradigms, performing experiments, acquiring and analyzing data, and producing figures for publication.

Structural Brain Imaging Techniques

Structural brain imaging techniques are used to resolve the anatomy of the brain in a living subject without physically penetrating the skull. These techniques can be used in combination with functional brain imaging techniques to correlate neural activity in specific anatomical regions with behavioral or cognitive functions. Structural techniques can also be used to measure anatomical changes that occur over time, such as a decrease in brain mass that occurs with aging or with the progression of disease. Most often, these techniques are used in clinical neuroscience and neurology to diagnose diseases such as tumors and vascular disorders.

Brain imaging technologies take advantage of the different composition of distinct brain regions and use these differences to form the basis of an image (Figure 1.1). Neural cell bodies contain many biomolecules, including proteins and carbohydrates. Axons and fiber tracts are relatively fatty due to the insulation provided by myelin. Cerebrospinal fluid (CSF) in the ventricles and surrounding the brain is essentially a saline solution. The microanatomy and composition of individual neural structures cause distinct regions of the brain to appear different when examined by the naked eye. For example, when looking at slices of the brain with the naked eye, brain tissue mostly composed of cell bodies appears gray compared with other areas, and thus is referred to as gray matter. Brain tissue mostly composed of axons and fiber tracts appears white, and thus is referred to as white matter. Often, the most informative structural images of the brain show the contrast between gray and white matter. Therefore, the ultimate goal of structural imaging technologies is to differentiate between proteins and carbohydrates (cell bodies), fat (axon tracts), and salt water (CSF), as this contrast reveals the most information about brain architecture.

Figure 1.1 The composition of the brain. (A) A microscopic view of a neuron. Each neuron is composed of a cell body, dendrites, and an axonal process. The cell bodies and dendrites are rich in proteins and carbohydrates. Axons are surrounded by a myelin insulation made of fats. (B) A macroscopic view of the brain. Gray matter is rich in cell bodies and therefore in proteins and carbohydrates. White matter is composed of axon tracts and is therefore rich in fatty myelin. CSF in the ventricles is a saline solution. To produce an image, brain imaging technologies must differentiate among proteins/carbohydrates, fats, and saline.

Until the early 1970s, there was no technology that could differentiate between these substances within the brain. Conventional X-ray technology is essentially useless for this purpose. During an X-ray procedure, an X-ray beam is passed through an object and then onto a photographic plate (Figure 1.2A). Each of the molecules through which the beam passes absorbs some of the radiation, so only the unabsorbed portions of the beam reach the photographic plate. X-ray photography is therefore only effective in characterizing internal structures that differ substantially from their surroundings in the degree to which they absorb X-rays, such as bone in flesh (Figure 1.2B). By the time an X-ray beam passes through the relatively soft consistency of the brain (not to mention the relatively hard consistency of the skull!), little information about individual brain structures can be discerned (Figure 1.2C). Therefore, in the 1960s and 1970s there was strong motivation to discover better ways of imaging the brain. The techniques described in the following sections represent 30–40 years worth of innovation in technology ultimately designed to show contrast within the soft tissue of the brain.

Figure 1.2 Standard X-ray technology alone cannot produce detailed images of the brain. (A) During an X-ray procedure, an X-ray beam is passed through an object and onto a photographic plate. Only the unabsorbed portions of the beam reach the plate, creating an image. (B) The contrast between the soft consistency of skin and muscle compared with the hard consistency of bone is sufficient to form an X-ray image. However, (C) the contrast between the soft consistency of different tissues within the brain is insufficient to form an X-ray image.

Cerebral Angiography

A cerebral angiogram is an enhanced X-ray that uses dyes to make up for the relatively poor soft-tissue contrast of conventional X-rays. A radio-opaque dye that absorbs X-rays better than surrounding tissue is injected into an artery that delivers blood to the brain. This substance heightens the contrast between the cerebral circulatory system and surrounding brain tissue during an X-ray (Figure 1.3A). Thus, the most prominent aspect of the central nervous system imaged in a cerebral angiogram is the brain vasculature. Angiograms can show vascular damage and indicate the presence of a tumor or aneurysm (Figure 1.3B).

Figure 1.3 Cerebral angiography. (A) A cerebral angiogram depicting the vasculature of the right hemisphere of the brain. (B) A cerebral angiogram indicating the presence of a brain aneurysm. (A, B: Reprinted from Nolte, J. and Angevine, J. B., (2007). The Human Brain in Photographs and Diagrams, 3rd ed. with permission from Mosby/Elsevier: Philadelphia. Courtesy of (A) Dr. Joachim F. Seeger and (B) Dr. Raymond F. Carmody.)

Computerized Tomography (CT)

Another method that improves upon conventional X-ray technology to image the brain and body is computerized tomography (the CT scan—sometimes also called computerized axial tomography, or CAT scan). A patient or subject lies with his or her head positioned in the center of a cylinder (Figure 1.4A). A narrow beam of X-rays is aimed through the person’s head and hits a detector on the opposite side. The beam and detector rotate in a slow arc, taking many